Excessive consumption of ethanol (EtOH) is associated with an increased incidence of certain infectious diseases and with suppression of a wide range of immunological functions. Innate immunity seems especially susceptible to the adverse effects of EtOH. The toll-like receptors (TLR) are a family of cell surface receptors that act to recognize molecular patterns unique to microbes and induce a signaling cascade that leads to the activation of a wide range of innate defense mechanisms. Published reports and preliminary data shown in this application indicate that EtOH suppresses responses to several ligands that are now known to act exclusively or predominantly through toll-like receptors. However, the characteristics (e.g., kinetics and EtOH dose-response relationships) and mechanisms of these effects are not known. Therefore, the work proposed here will test the following hypothesis: EtOH is an inhibitor of innate defense mechanisms activated through toll-like receptors (TLR) 3 and 4, it acts by suppressing events in the TLR signaling cascade and thus decreasing production of key cytokines, chemokines, and inducible nitric oxide synthase, and this will cause decreased resistance to bacterial peritonitis and septicemia. This will be accomplished by pursuing three specific aims: 1) Determine the effects of acute EtOH administration with regard to the induction of cytokine, chemokine, and inducible nitric oxide synthase (iNOS) production induced by a ligand for TLR 3 (poly I:C) and TLR 4 (bacterial lipopolysaccharide, LPS). 2) Identify TLR 3 and TLR 4-induced signaling events that are affected by EtOH and determine their role in altering cytokine, chemokine, and iNOS production. 3) Investigate the effects of acute EtOH administration on resistance to E. coli peritonitis and septicemia and the involvement of cytokines and signaling components in these effects. The results will indicate the extent to which EtOH-mediated changes in TLR signaling contribute to immunologically relevant effects. An important and unique feature of these studies is that the EtOH exposure and TLR stimulation will be done in vivo (in mice). Studies with cell lines have been critical in elucidating TLR signaling pathways, but macrophage responses are remarkably different in vitro and in vivo. This project represents a novel and innovative approach to bridge the gap between molecular causation (Aims 1 and 2) and modeling at the whole animal level (Aim 3) and we expect it to have significant impact in both immunology and systems biology.